Overview - What is liquid biopsy?
DNA and RNA fragments are released into body fluids such as blood and urine via various physiological processes. When released into the bloodstream, the so called cell-free DNA (cfDNA) and cell-free RNA (cfRNA) are collectively referred to as circulating nucleic acids in plasma or serum (CNAPS). The majority of cfDNA in circulation originate from passive release by cells that undergo cell death mechanisms such as apoptosis and necrosis, and from active secretion by living cells. The size of most cfDNA fragments ranges between 180 and 200 bp, but fragments as large as 300 bp have also been reported (Figure 1). The half-life of cell-free DNA is less than two hours before it is filtered out of the blood circulation by the spleen, liver and kidneys.
Similarly to healthy cells, primary and metastatic cancer cells also release DNA fragments into the bloodstream. The resulting circulating tumour DNA (ctDNA) is characterised by genetic defects such as point mutations, chromosomal rearrangements, abnormal epigenetic patterns and copy number aberrations. The genetic defects found in ctDNA are identical to those found in the tumour source and they are completely absent from normal circulating DNA. Since ctDNA is representative of the entire tumour genome, the analysis of tumour DNA from fluid samples is frequently referred to as whole body biopsy or “liquid biopsy”.
Figure 1: Tumours can secrete fragments of DNA into the blood circulation (ctDNA). ctDNA is found in plasma. A liquid biopsy using blood samples obtained from cancer patients can be performed to detect ctDNA and to identify specific mutations that may have prognostic or therapeutic implications.
Other components of blood that could act as tumour surrogates include circulating tumour cells (CTC), circulating miRNA, exosomes and circulating vesicles.
Applications - What are the advantages of liquid biopsies?
The use of cfDNA holds great diagnostic potential for various clinical scenarios. Currently, the use of cfDNA as a non-invasive tumour tracker for cancer screening and diagnostics is captivating most of the attention of the life science community.
Cell-free DNA as a cancer biomarker
The ability to analyse ctDNA that has been isolated from a simple blood draw provides rapid, affordable and non-invasive access to versatile biomarkers that accurately reflect changes in the tumour of origin. These characteristics of ctDNA make it attractive to a number of different areas in oncology.
The clinical utility of ctDNA analysis for cancer detection includes:
- Assessment of molecular heterogeneity
- Identification of specific genomic alterations to guide treatment selection
- Monitoring of tumour burden
- Early detection of therapy resistance
- Detection of minimal residual disease
- Evaluation of dissemination and recurrence risks
- Understanding mechanisms of resistance
Liquid biopsy as a complement to tissue biopsy
Tissue biopsy is currently the standard of care for the molecular diagnosis of cancer. The conventional approach involves examination of tumour tissue by removing cells through a small needle or histological examination of a biopsy or surgical excision specimen. Molecular diagnosis of DNA mutations is then performed directly on fresh tissue samples or often on formalin-fixed paraffin embedded tissue (FFPE) material. Despite the informative nature of solid tumour biopsies, tumour heterogeneity and clonal evolution are significant challenges for the design of effective treatment strategies based on tissue biopsies alone. Molecularly targeted, personalised cancer therapies depend on serial monitoring of cancer genetics. Performing consecutive biopsies to capture a tumour’s spatial and temporal heterogeneity during tumour evolution is often difficult, risky, expensive, and often times unachievable. Therefore, the need for novel approaches that could efficiently detect tumour heterogeneity in the course of cancer system treatments has arisen.
Recent progress in genomic analysis of blood samples for ctDNA made a real-time, affordable, and non-invasive liquid biopsy approach for cancer detection and monitoring possible. This novel diagnostics method provides important complementary information on therapeutic targets and drug resistance mechanisms in patients who suffer from cancer.
Other applications of cell-free DNA
Analysis of cfDNA analysis can be beneficial for other medical fields besides oncology. Analysis of cell-free foetal DNA is an established method for non-invasive prenatal testing (NIPT). Examination of foetal DNA can uncover point mutations and aneuploidy that are responsible for genetic disorders, as early as seven weeks following conception. In Europe, foetal DNA found in circulation of expecting mothers has been used for prenatal screening since 2012.
The state of the cfDNA that is extracted from plasma can also serve as an indicator for pathogen infections, neurodegenerative, autoimmune and cardiovascular disease, or as an early warning sign of organ rejection.
Workflow – How does liquid biopsy work?
Currently, blood-based cancer testing involves the assessment of biomarkers, most commonly cfDNA, circulating tumour cells (CTCs), or exosomes. Other prognostic and predictive markers for early-stage cancer development include cell-free RNA (cfRNA) fragments, methylated ctDNA and miRNA.
ctDNA is a particularly promising circulating biomarker for risk assessment in cancer patients due to the simplicity of obtaining plasma DNA, the cost-efficiency of the process and the high specificity and sensitivity of the resulting data. As one of the most established techniques, liquid biopsies based on ctDNA analysis are closest to the implementation in clinical settings. Here, several strategies exist for exploring tumour DNA found in plasma. Whole genome sequencing or whole exome sequencing approaches provide the most comprehensive view of tumour-associated mutations, but with lower sensitivity. Targeted approaches that are focused on specific genomic aberrations offer higher sensitivity, but no access to molecular information outside of the targeted regions of interest. Mutations that are most often assessed using targeted approaches for liquid biopsies include EGFR, PIK3CA, KRAS, BRAF, TP53, HER2, GNA11, KIT, MAP2K1, NRAS and others.
Next generation sequencing of ctDNA that has been isolated from blood follows a similar workflow regardless of whether specific targets or the whole genome is investigated. The extraction of cfDNA from plasma is one of the most important steps and must be performed in optimal conditions to ensure that downstream processing is successful.
Subsequently to DNA extraction, library preparation protocols that are optimised for low-input DNA are followed. Next generation sequencing is then performed on the appropriate platform, and the resulting reads are subjected to thorough BioIT analysis. The final data is then delivered to a cancer researcher for a scientific interpretation or to a physician for a medical conclusion (Figure 2).
Figure 2: Workflow example for next generation sequencing of ctDNA
Scientific expertise: liquid biopsy
Eurofins Genomics offers access to liquid biopsy-based assays with unprecedented flexibility for any stage or any project of blood-based tumour characterisation.
Over the years, Eurofins Genomics has developed a proprietary protocol for highly efficient extraction of miniscule amounts of cfDNA from plasma. The DNA is either sequenced in specialised next generation sequencing workflows or amplified using the latest droplet digital PCR (ddPCR) technologies. Additionally, established approaches for target enrichment and variant analysis are included in the exome sequencing service.
All Eurofins Genomics services offer sophisticated logistics, transparent LIMS-controlled sample and data processing, followed by convenient data delivery. The Eurofins Genomics Oncology solutions are processed in our own labs.
Related products to liquid biopsy
Are you ready to implement liquid biopsies in your cancer research studies? Find out how you can help advance personalised medicine through improved cancer detection and screening with our liquid biopsy service line.
Our Oncology Solutions have complementary products that are fully compatible with tissue or FFPE samples. The joint use of these services makes it possible to compare investigations of DNA from matched tumour tissue and plasma samples.
Eurofins Genomics has also great expertise in Sanger sequencing, oligo and gene synthesis, as well as in NGS that all converges into a comprehensive product portfolio? Check out the Sanger sequencing serives, DNA & RNA Oligonucleotides, and Gene Synthesis & Molecular Biology services.